Experiment:

Extraction and Estimation of Proteins from plant sources

Extraction of tested material:

Small piece of a solid material (soaked peas, beans, potato tuber) can be ground in
water. Squeezed this material with filter paper. Liquid solution is then used for protein test
after centrifugation.

Tests for Protein

1) Ninhydrin Test:
Principle:
Ninhydrin is a chemical used to detect primary and secondary amines. Amino acids also react
with ninhydrin at pH=4. The reduction product obtained from ninhydrin then reacts with NH3
and excess ninhydrin to yield a blue coloured substance. This reaction provides an extremely
sensitive test for amino acids.

Requirements
§ Test solution

§ Ninhydrin reagent

§ Water bath

§ Dry test tubes

§ Pipettes

Procedure
1. Take 1 ml test solution in dry test tube and 1 ml distilled water in another tube as a
control.

2. Pour few drops of ninhydrin reagent in both the test tubes

3. Keep the test tubes in water bath for 5 minutes

Result:
Appearance of blue or violet color indicates the presence of protein
 2) Biuret test
Principle:
Biuret test is a general test for compounds having a peptide bond. Biuret is a
compound formed by heating urea to 180° C. When biuret is treated with dilute copper sulfate
in alkaline condition, a purple colored compound is formed. This is the basis of biuret test
widely used for identification of proteins and amino acids.

Requirements:
§ Test solution

§ Biuret reagent (CuSO4, NaOH)

§ Dry test tubes

§ Pipettes

Procedure:
1) Take 1 ml of test solutions in dry test tubes and in another tube take 1 ml distilled
water as control.
2) Add 1 ml of biuret reagent to all test tubes, mix well.

Result:
Appearance of violet color indicates the presence of protein